Comprehensive Study Notes: Tumor Immunology and Immunotherapy in Veterinary Medicine

Session Learning Objectives

• Explain the various mechanisms by which the immune system can eliminate tumors.
• Explain how tumor cells may evade or escape the immune system.
• List specific conditions that render cancer cells readily visible to immune cells.
• List methods by which the immune system can be actively or passively activated against cancer.
• Identify specific cancers that affect the immune cells themselves.
• Define immunotherapy and explain the mechanisms of the following approaches:
      * Checkpoint inhibition.
      * Immune cell therapy.
      * Active immunization.

Mechanisms of Tumor Development and Immune Surveillance

• Tumor Development Traits: Tumor cells possess distinct capabilities allowing for unregulated growth:
      * The ability to replicate indefinitely and rapidly, free of normal physiological regulation.
      * The ability to defy programmed cell death (apoptosis).
      * Growth that does not require external growth factors.
      * The capability to metastasize to distant tissues.
      * Characterized by genomic instability and high rates of mutation.
      * Development involves multiple mutations in regulatory genes.
• Immune Surveillance and Control: The body can develop immunity to tumors. The immune system is theoretically in a state of constant surveillance, editing, and control.
• Prerequisites for Immune Response: Certain conditions must be met for the immune system to recognize a tumor:
      * Virus-induced tumors: May produce foreign viral proteins that the immune system recognizes.
      * Inflammation-triggering tumors: Local inflammation can trigger the activation of immune cells.
      * High Mutation Rates: Rapidly mutating tumors generate "neoantigens" that appear foreign.
• The Recognition Complexity: Note that immunosuppressed individuals are not at a significantly higher risk for common cancers, illustrating that immune recognition of tumors is a complex process rather than a simple failure of surveillance.

Major Tumor Antigens and Visibility

• Tumor visibility to the immune system depends on the expression of specific antigens:
      * Mutational Antigens: Arise from genetic changes leading to altered surface proteins.
      * Inappropriate Expression: Antigens typically found in embryos but expressed inappropriately in adults, such as Carcino Embryonic Antigen ($CEA$) and $\alpha$-fetoprotein ($AFP$).
      * Overexpression: Proteins present on normal cells but found in much higher quantities on tumor cells, such as $EGFR$, $HER2$, and $PSA$.

Components of Immune Response to Tumors

• Macrophages and Dendritic Cells (DCs): Engage in direct attack or present tumor-derived antigens to the adaptive immune system.
• Antibody-Dependent Cellular Cytotoxicity (ADCC): Occurs if antibodies are produced against surface antigens. This is particularly effective for blood cancers because malignant cells are readily accessible to antibodies.
• Natural Killer (NK) Cells: Targeted especially against cells with abnormal surface compositions or those that lack Major Histocompatibility Complex ($MHC$) molecules.
• CD8+ Cytotoxic T Lymphocytes (CTL): Attack cancer cells that display matching $MHC$ molecules.
• Cytokine Signaling (Figure 33-3):
      * Macrophages produce $IL-12$, $TNF-\alpha$, and $IFN-\gamma$.
      * T cells produce $IFN-\gamma$ and $IL-2$.
      * NK cells produce $IFN-\gamma$.
      * $IL-2$ and $IL-12$ are critical for stimulating the activity of these cells against the tumor.

Mechanisms of Tumor Evasion (Failure of Immunity)

• The "Self" Barrier: Because tumor cells originate from "self" tissues, they often lack traditional "foreign" markers.
• Evasion Strategies once the "Self" Barrier is Broken:
      * Down-regulation of MHC: Tumors stop expressing $MHC$ and co-stimulatory signals to become "invisible" to T-cells.
      * Antigen Shedding: Tumors may shed or limit the surface expression of potential antigens to appear like normal cells.
      * Immunosuppressive Cytokines: Release of factors like $TGF-\beta$, $IL-4$, and $IL-10$ which act as "No Danger" signals to inhibit immune action.
      * Active Defense (FasL): Enhance T-cell death through mechanisms such as Fas Ligand ($FasL$).
      * Regulatory T-cell (Treg) Recruitment: Attract $Treg$ cells that actively suppress the activity of other immune cells.
      * Microenvironment Exclusion: Creating "cold" tumors by physically or chemically excluding immune cells from the tumor microenvironment.

General Immunotherapy Approaches

• Immune Stimulants:
      * Cytokines: Including $IFN$, $TNF$, $IL-2$, and $GM-CSF$.
      * Bacterial Products: Such as Bacillus Calmette-Guérin ($BCG$).
• Immunotoxins: Antibodies directed against tumors, either used alone or conjugated to a toxin.
• Lymphokine Activated Killers (LAKs): Lymphocytes are removed from the patient, activated in vitro, and injected back into the patient.
• Checkpoint Inhibition: Stimulating T-cells by blocking inhibitory signals (checkpoint blockade) that normally stop T-cells from indefinite proliferation.
• T-cell Therapy (CAR cells): Engineering T-cells ex vivo (in labs) before returning them to the patient (Chimeric Antigen Receptor T-cell therapy).
• Active Immunization: Therapeutic vaccines (mostly experimental). The $HPV$ cervical cancer vaccine is a preventive vaccine targeting the virus rather than an existing tumor.

Immunotherapy in Veterinary Medicine

• Current State: Progress in veterinary medicine lags behind human medicine due to fewer identified targetable antigens and higher costs.
• Approved Veterinary Vaccines:
      * Oncept® (Merial): The first USDA-approved commercial tumor vaccine for canines. It targets the (human) tyrosinase protein in melanoma by injecting human or murine genes. Its efficacy under various conditions remains controversial.
• Monoclonal Antibodies (mAbs) for Dogs:
      * Blontress®: Targeted against $CD-20$ for B-cell lymphoma.
      * Tactress®: Targeted against $CD-56$ for T-cell lymphoma.
• Comparative Costs (Human vs. Potential Vet Application):
      * $Nivolumab$: approximately $\$103,220$.
      * $Ipilimumab$: approximately $\$158,252$.
      * $Kymriah$ and $Yescarta$ (CAR-T): between $\$373,000$ and $\$475,000$ per patient.
• Research and Clinical Trials:
      * Yale ErbB Vaccine: A peptide vaccine targeting $EGFR$ and $HER2$ ($EGFR$/$HER2$). It elicits antibodies that bind both human and canine proteins, inhibiting tumor growth and triggering the homing of $CD8+$ T cells to solid tumors ($Colorectal$, $Breast$, $Osteosarcoma$).
      * Autologous Lymphocytes: Repeated infusions of cells expanded ex vivo with anti-$CD3$ mAb and human cytokines $IL-2$ and $IL-21$ improved survival in dogs with lymphoma (median $167$ days vs. $392$ days) according to O’Connor et al $2012$.

Specific Immunologically Relevant Animal Tumors

• Injection Site-Associated Sarcomas (Cats):
      * Incidence: Approximately $1/10,000$ or less cat vaccinations.
      * Pathogenesis: Linked to local chronic irritation from adjuvants, antibiotics, sutures, or cotton pieces. Involves the pro-inflammatory cytokine $IL-23$.
      * Guidelines: Rabies vaccine in the Right limb, Leukemia vaccine in the Left limb.
• Transmissible Venereal Tumor (TVT/CTVT):
      * Transmission: Physical implantation of cells during copulation. Clonal origin dating back thousands of years.
      * Immune Response: Most dogs clear the cells via antibodies and T-cells. Aggressive tumors down-regulate $MHC-I$.
• Bovine Lymphosarcoma (Bovine Viral Leukosis):
      * Prevalence: In the USA, over $45\%$ of dairy and over $10\%$ of beef cattle are infected.
      * Etiology: Bovine Leukemia Virus ($BLV$), a retrovirus transmitted via infected lymphocytes (needles, flies, colostrum).
      * Clinical Signs: Only $5\%$ develop clinical disease (lymphocytosis). Causes immunosuppression via reduced T-cell counts. No vaccine or treatment exists.
• Marek Disease (MD) vs. Avian Lymphoid Leukosis (ALL):
      * MD: Herpes virus-induced T-cell tumor. Symptoms include enlargement of the sciatic nerve and sacral plexus. Vaccine is available.
      * ALL: B-cell lymphoma induced by Avian Leukosis Viruses (retrovirus). No vaccine available.
• Canine Lymphomas:
      * The most common hematopoietic tumor in dogs.
      * Primarily large B-cell tumors, though T-cells can also be malignant. Affects skin, lymph nodes, thymus, spleen, GI, and mediastinum.

Questions and Discussion

• Pre-session Question 1: How may tumor cells evade the immune system?
      * Response: All of the above (Produce self antigens, suppress CMI activation, suppress $MHC$, activate regulatory T-cells).
• Feline Vaccination Question: What tumor type is a cat predisposed to developing at vaccination sites?
      * Response: Fibrosarcoma.
• Clinical Case: Fine needle aspirate, popliteal lymph node, Lab Retriever, Wright Giemsa stain, $500X$:
      * Question: What cells are most prominent? What is different about them? Why could the immune system not control these?
      * Context: This refers to the heterogeneity and malignant transformation of lymphocytes in canine lymphoma.
• Checkpoint/Test Yourself Checklist:
      * Immune activation by vaccination: Possible for Marek disease (Herpes), TVT (Cells), and Cutaneous warts (Papilloma virus).
      * Common features of ALL and BVL: Both are retrovirus-induced and affect lymphocyte populations.
      * Immunosuppression in lymphocytic cancer: Occurs because the malignant cells are non-functional or actively suppress healthy immune populations.

Summary Points

• Tumors are altered "self" but can be recognized via neoantigens or viral antigens.
• Major players: Macrophages, NK cells ($non-MHC$ targets), and $CD8+$ T-cells ($MHC$ targets).
• Success of tumors depends on mechanisms like $MHC$ down-regulation, $Treg$ expansion, and T-cell killing.
• Immunotherapy methods include cytokines, checkpoint inhibition, and ex vivo cell activation.
• Veterinary-specific conditions include Injection-site sarcomas, TVT, BVL, MD, and ALL.